Antimicrobial peptides against Listeria monocytogenes

ABSTRACT

The present invention relates to the use of at least one peptide having sequence VRLIVX 1 VRIX 2 RR (SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4), wherein X1 is selected from A or K and X 2  is selected from W and K, as a bactericidal antimicrobial agent against  Listeria monocytogenes.

This application is a U.S. national stage of PCT/EP2018/069304 filed on16 Jul. 2018, which claims priority to and the benefit of ItalianApplication No. 102017000080068 filed on 14 Jul. 2017, the contents ofwhich are incorporated herein by reference in their entireties.

DESCRIPTION

Sequence listing ASCII file Seql-000001, created on Jan. 9, 2020 and ofsize of 37,300 bites is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to peptides having bactericidal activitytowards Listeria monocytogenes and to their use as antimicrobial agentsor in the treatment of listeriosis.

The invention also relates to products having the above peptides coatedor immobilised on their surface.

BACKGROUND ART

The consumer's attention to the possible health effects related to foodquality is having a major impact on the food processing and preservationindustries. Compared to the past, today the consumer is increasinglyaware and attentive to the quantity of chemical preservatives present infood and to the effectiveness of both conventional methods andinnovative preservation technologies to extend durability and improvethe safety of a wide range of food products. There are severalmicroorganisms recognized today as responsible for food infections, i.e.related to the consumption of food, among these there is certainlyListeria monocytogenes, a Gram-positive bacterium, pathogen ofListeriosis. In fact, this may contaminate a wide variety of foods,usually raw, such as not well cooked meat, raw vegetables, fishproducts, cheese prepared from unpasteurized milk or in ready-to-usefood products that are industrially processed and require preservationat low temperatures. Once ingested, Listeria monocytogenes can crossintestinal, placental and cerebral barriers causing infections withdifferent clinical features, including serious pathological events suchas spontaneous abortions, meningoencephalitis, septicemia andgastroenteritis. Food-originated Listeriosis is a rather rare butserious disease, with a mortality rate of 20-30%, comparable to that ofother food diseases such as salmonellosis. In relation to its incidenceand severity, the economic and social impact of listeriosis isconsidered among the highest among foodborne diseases (Barbuddhe S B andChakraborty T., 2009, Curr Top Microbiol Immunol 337:173-95; Barbuddhe SB et al., 2012, Int J Food Microbiol 154(3):113-8).

The contamination of food by this microorganism is very widespread anddifficult to fight as it is able to adapt to different food storage andproduction processes conditions. In fact, Listeria monocytogenes cangrow and reproduce at different pH conditions and at temperaturesranging from 0 to 45° C., thus persisting in the environment as well asin processed, stored and refrigerated foods. It has also been shown thatListeria monocytogenes is capable of resisting cleaning, sanitizing,drying and UV ray processes, thus increasing the likelihood ofenvironmental contamination (Mullapudi S et al, 2008, Appl EnvironMicrobiol 74: 1464-8, Saá Ibusquiza P et al, 2011, Food Microbiol28(3):418-25) Listeria monocytogenes infections have traditionally beentreated by administration of penicillins or carbapenems (Espaze E P andReynaud A E, 1988, Infection 16 Suppl 2: S160-S164, Troxler R et al,2000, Clin Microbiol Infect 6: 525-535). However, in recent yearsseveral Listeria strains have been identified, showing multipleresistance to antibiotics, including those of election for the treatmentof the infection (Walsh D et al, 2001, J Appl Microbiol 90: 517-522,Prazac A M et al, J Food Prot. 65: 1796-1799, Charpentier E et al, 1995,J Infect Dis 172: 277-281). In particular, the most represented strainin clinical listeriosis isolates is the serotype 4b (Gilot P et al,1996, J Clinical Microbiol 34(4): 1007-1010, Mammina C et al, 2009, JClinical Microbiol 47(9): 2925-2930). This strain is more virulent thanthe reference ATCC (American Type Culture Collection) or NTCC (NationalType Culture Collection) strain, available on the market. Moreover, theserotype ½c, which is one of the most frequently isolated from food, hasrevealed a specific resistance to penicillin G and ampicillin (Ayaz etal, 2010, J. Food protection 73: 967-972).

In view of the above, the need is felt to identify new molecules thatallow an effective action of prevention and/or treatment of bothcontamination of products, in particular food, by Listeriamonocytogenes, and listeriosis infections, in particular in the case ofantibiotic-resistant strains.

In the last decade, the existence of natural peptides (calledAntimicrobial Peptides or AMPs) having various and diverse antimicrobialactivities has been recognized (De Smet K and Contreras R, 2005,Biotechnol Lett 18:1337-47). In humans and other mammals, these peptideshave been identified as essential components of innate immunity,contributing to the first line of defense against infections (HenzlerWildman K A et al, 2003, Biochemistry 42(21):6545-58; Bals R, 2000,Respir Res. 1(3):141-50, Agerberth B et al, 1999, Am J Respir Crit CareMed 160: 283-290).

The specific antimicrobial activity and the selectivity of AMPs dependson their chemical-physical properties such as: amphipathiccharacteristics, net charge, charge angle, total hydrophobicity andconformational flexibility (Teixeira V et al, 2012, Prog Lipid Res.51(2):149-77, 2012, Palmieri G et al, 2016, Food Chem 211:546-54). Inparticular, the peptides show a structure-activity relationship forantibiofilm activity different from that requires for bactericidalactivity against planktonic cells (De la Fuente-Nuñez C, 2014, PLoSPathog 10(5):e1004152).

In recent years, numerous peptides have been identified as potential newantimicrobial agents through various techniques such as screening andtesting of natural host defence peptides, in silico analysis orscreening of peptide libraries. Some of these peptides have shown anactivity against bacterial biofilm formation, at concentrations oftenbelow their minimum inhibitory concentration (MIC) against the samebacterial cells in planktonic form. It has also been pointed out thatthe antimicrobial activity of the identified peptides does not appear inany way related to a bactericidal activity because thestructure/function relationship of the peptides is different for thebactericidal or antibiofilm activities (Overhage J et al 2008, InfectImmun 76(9):4176-82, Amer L S et al, 2010, Biochem Biophys Res Commun396(2):246-51, Pompilio et al, 2011, Peptides 32(9):1807-14, De laFuente-Nuñez C et al, 2012, Antimicrob Agents Chemother 56(5):2696-704,De la Fuente-Nunez C et al, 2014, PLoS Pathog 10(5):e1004152).

In particular, WO2015038339 describes 749 peptides having a sequence of7-12 aminoacids, which are indicated as having a very specificantibiofilm and/or immunomodulatory activity. In this context, thepeptide named IDR-1018, having sequence VRLIVAVRIWRR, (SEQ ID NO: 1),the peptide named IDR-1018-K6 or 2001, having sequence VRLIVKVRIWRR (SEQID NO: 3), and the peptide named IDR-1018-K10, having sequenceVRLIVAVRIKRR (SEQ ID NO: 2) are disclosed. The antibiofilm activity ofthese peptides is demonstrated against a number of differentmicroorganisms such as Pseudomonas aeruginosa, Klebsiella pneumoniae,Staphylococcus aureus, E. coli, and Acinetobacter baumannii, Salmonellaenterica ssp. Typhimurium and Burkholderia cenocepacia or Staphylococcusaureus and Pseudomonas aeruginosa.

The antibiofilm activity of peptide 1018 against biofilms formed byPseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, E.coli, Acinetobacter baumannii and Burkholderia cenocepacia is alsoreported by the same authors in PLoS Pathog 10(5):e1004152.

The above documents disclose that the peptides show a specificantibiofilm activity that is independent of the bactericidal activityagainst cells in planktonic form and do not show any activity onplanktonic cells at the concentrations tested.

In view of the above, in WO2015038339 it is suggested to combine thepeptides with antibiotics that kill bacteria released from the biofilmonce this is disrupted.

Bacteria belonging to the Listeria genus show very peculiarcharacteristics and there are numerous evidences demonstrating thatpeptides capable of an efficient growth inhibition of other bacteriashow instead a scarce activity against Listeria monocytogenes (GravesenA et al 2002, Microbiology 148:2361-2369, Gravesen A et al 2001, MicrobDrug Resist 7:127-135, Rasch M and Knøchel S 1998, Lett Appl Microbiol27:275-278).

Several studies have shown that, compared to other microorganisms suchas Pseudomonas or Staphylococcus, the formation of biofilm is lesscritical in infections caused by Listeria since this pathogen has alower capacity to form biofilms (Renier S et al, 2011, Environ Microbiol13: 835-50, Djordjevic D et al, 2002, Appl Environ Microbiol 68:2950-2958, Borucki M K et al, 2003, Appl Environ Microbiol 69:7336-7342, Harvey J et al, 2007, Food Microbiol 24: 380-92). Morerecently, assays carried out on a broad panel of Listeria monocytogenesstrains isolated from clinical and food sources have shown an extremevariability and a poor tendency to the formation of biofilms (Doijad S Pet al, 2015, PlosOne 10(9): e0137046, Borucki M K et al, 2003, ApplEnviron Microbiol 69: 7336-7342). In particular, none of the strainsfrom animal and human clinical cases showed a strong capacity of biofilmformation (Doijad S P et al, 2015, PlosOne 10(9):e0137046). In addition,the films formed by Listeria and the mechanisms by which the bacteriaadhere to surfaces appear to be completely different from those of theother bacteria. In particular, Listeria does not secrete a sufficientamount of extracellular matrix polysaccharides to producethree-dimensional films as the bacteria listed above, but adhere to thesurfaces through hydrophobic interactions forming bidimensional films(Silva E P et al, 2013 Appl Microbiol Biotechnol 97: 957-68, Doijad S Pet al, 2015, PlosOne 10(9): e0137046).

In the case of Listeria, in order to effectively prevent or eliminatecontamination by this microorganism, an antimicrobial agent having astrong bactericidal activity against planktonic cells is required. Theactual treatment of Listeria contamination or infection with antibioticsfinds a great limitation in the resistance developed by many strains ofthese bacteria. An ever-increasing number of Listeria strains appears tohave a worrisome reduced susceptibility to various antibiotics such asampicillins, penicillins and gentamicins (Facinelli B et al, 1991,Lancet 338: 1272, Safdar A and Armstrong D, 2003, J Clin Microbiol 41:483-485, Popowska M et al, 2006, Pol J Microbiol 55: 279-288).

It is therefore strongly felt the need of identifying new antimicrobialagents against Listeria infections that show a strong bactericidalactivity, optionally associated with an ability to also inhibit biofilmformation, especially against antibiotic resistant strains.

Molecules capable of performing both an effective bactericidal andantibiofilm activity against Listeria strains can significantly increasethe levels of food safety, also ensuring better therapy and prophylaxisof listeriosis.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that the peptides IDR-1018and IDR-1018-K6, the latter hereinafter referred to as Bac-amp1, andpeptides exhibiting a sequence having the same features of length,basicity, net charge and hydrophobicity exhibit a bactericidal activityagainst Listeria monocytogenes with a MIC below 10 μM and a weakantibiofilm activity, with a MIC between 25 and 50 μM.

The biological activity observed for these peptides against Listeria iscompletely unexpected. In fact, this is significantly different fromthat disclosed by the prior art discussed above for the same peptidesagainst other microorganisms, which showed an antibiofilm activity atlow concentrations of peptide, at which no activity against planktoniccells was identified.

The aforesaid peptides represent excellent candidates able to meet thefood safety requirements, reducing the levels of contamination uponconsumption, and improving the prevention and prophylaxis fromlisteriosis, caused by the pathogenic agent Listeria monocytogenes.

Accordingly, a first object of the present invention is the use of atleast one peptide, having sequence VRLIVAVRIWRR, VRLIVAVRIKRR,VRLIVKVRIWRR, VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4), or a salt or solvate thereof, as an antimicrobial agentfor the prevention and/or treatment (removal) of contamination of aproduct, preferably a food, a material, an object, or a surface byListeria monocytogenes.

Furthermore, the present inventors have found that the peptidesaccording to the invention show a high conformational stability even atextreme pH values (pH 1.0 or 11.0). In particular, the peptides of theinvention such as for example Bac-amp1, show a greater conformationalstability at pH 11.0; on the contrary, the peptides of the inventionsuch as for example IDR-1018, have a higher conformational stability atpH 1.0. These properties make it possible to use these peptides, eitherindividually or in combination, in acidic or basic environments, such asin the presence of chemical compounds used in sanitizing formulations.This allows the development of sanitizing eco-friendly bio-formulationswith a lower concentration of chemical substances, acidic or stronglyalkaline and, as a consequence, a reduced environmental impact.

Furthermore, an association of the two peptides indicated above isparticularly advantageous, since it allows obtaining antimicrobialcompositions effective within a very broad pH range.

Therefore, a second object of the invention is an antimicrobialcomposition comprising a mixture of at least one peptide having sequenceVRLIVAVRIWRR, VRLIVAVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2) and at least onepeptide having sequence VRLIVKVRIWRR, VRLIVKVRIKRR (SEQ ID NO: 3, SEQ IDNO: 4), preferably a mixture between the peptide having sequenceVRLIVAVRIWRR (SEQ ID NO: 1) and the peptide having sequence VRLIVKVRIWRR(SEQ ID NO: 3).

A third object of the invention is a peptide having sequenceVRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), or a pharmaceuticallyacceptable salt or solvate thereof, for use in the treatment of aninfection from Listeria monocytogenes in a subject, preferably for usein the treatment of listeriosis.

A fourth object of the invention is a method for the treatment of aninfection from Listeria monocytogenes in a subject, preferablylisteriosis, by administering a peptide or a mixture of peptides havingsequence VRLIVAVRIWRR, VRLIVAVRIKRR, VRLIVKVRIWRR, VRLIVKVRIKRR (SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), or a pharmaceuticallyacceptable salt or solvate thereof.

A fifth object of the invention is a pharmaceutical formulationcomprising at least one peptide having sequence VRLIVAVRIWRR, orVRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: and SEQ ID NO: 4), or a pharmaceutically acceptable saltor solvate thereof.

According to a preferred embodiment, said at least one peptide is amixture between at least one peptide having sequence VRLIVAVRIWRR, orVRLIVAVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2) and at least one peptidehaving sequence VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO:4), preferably a mixture between the peptide having sequenceVRLIVAVRIWRR (SEQ ID NO: 1) and the peptide having sequence VRLIVKVRIWRR(SEQ ID NO: 3).

A sixth object of the invention is a coating composition comprising atleast one peptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR, orVRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:and SEQ ID NO: 4).

A seventh object of the present invention is a product having at leastone surface covered with a coating adherent to said surface comprisingat least one peptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR, orVRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:and SEQ ID NO: 4).

The present inventors have also surprisingly found that when theN-terminal amino group of the above peptides are linked covalently tothe surface of a product, the bactericidal activity of the free peptidesis maintained.

Accordingly, an eighth object of the invention is a product having atleast one surface, covalently linked to at least one peptide havingsequence VRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR(SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: and SEQ ID NO: 4).

A ninth object of the invention is a liquid antimicrobial compositioncomprising the nanoparticles comprising, covalently linked to theirsurface, at least one peptide having sequence VRLIVAVRIWRR, orVRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4).

A tenth object of the invention is the use of nanoparticles comprising,covalently linked to their surface, at least one peptide having sequenceVRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4) as an antimicrobialagent for the prevention or treatment of contamination of a product orsurface by bacteria.

An eleventh object of the invention is a method for the treatment of abacterial infection in a subject, by administering nanoparticlescomprising, covalently linked to their surface, at least one peptidehaving sequence VRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, orVRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4).

A twelfth object of the invention is a pharmaceutical compositioncomprising nanoparticles comprising, covalently linked to their surface,at least one peptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR, orVRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4), optionally in combination with pharmaceuticallyacceptable excipients or carriers.

FIGURES

FIG. 1 and FIG. 2 show the Circular Dichroism (CD) spectra of theBac-amp1 and IDR1018 peptide, respectively, obtained in the presence of10 mM SDS under different temperature conditions, as described inExample 2a.

FIG. 3 and FIG. 4 show the Circular Dichroism (CD) spectra of theBac-amp1 and IDR1018 peptide, respectively, obtained in the presence orabsence (SDS) of 10 mM SDS at different pH conditions, as described inExample 2b. The diagrams show in particular the average molarellipticity values per residue×10⁻³, expressed in degrees cm²/dmol, asthe wavelength varies.

The folding kinetics of the Bac-amp1 peptide was monitored for 24 h at25° C. by CD spectroscopy (C) and fluorescence (D) techniques after theaddition of SDS at a final 10 mM concentration.

FIG. 5 shows a chromatogram obtained by RP-HPLC chromatography performedon samples of the IDR-1018 or Bac-amp1 peptide incubated in mozzarellabrine for 24 h at 4° C. as described in Example 2c. The diagram showsthe absorbance values at 220 nm as a function of the volume of eluate,measured in ml.

FIG. 6 shows the dose-response curve obtained with the Bac-amp-1peptides against S. aureus, S. thyphimurium, L. monocytogenes, L.monocytogenes and L. monocytogenes, expressed as % of colony formingunits (CFU) observed on plates seeded with the bacterial cultures uponincubation in the presence of different peptide concentrations, asdescribed in Example 3, with respect to the corresponding control plateswithout peptide.

FIG. 7 and FIG. 8 show the box-plot graphs, which are a graphicalrepresentation used to describe the distribution of a sample by means ofsimple dispersion and position indices, obtained by using the GraphpadPrism software. The figures show the absorbance values at 492 nmmeasured after staining with crystal violet of steel discs incubatedwith a standardized inoculum of L. monocytogenes for 72 h and at 37° C.in absence (Ctrl) or in the presence of different concentrations ofBac-amp1 or IDR-1018 peptide, respectively, as described in Example 4a.

FIG. 9 shows the images at different magnifications obtained with thescanning electron microscope (SEM) of steel discs treated with astandardized inoculum of L. monocytogenes in the presence of theBac-amp1 peptide (FIG. 9a , magnification×1000; FIG. 9c ,magnification×5000) at the concentration 50 μM, or only with controlmedium (FIG. 9b , magnification×1000; FIG. 9d , magnification×5000), asdescribed in Example 4b.

FIG. 10 shows the images at different SEM magnifications of steel discstreated with standardized inoculum of L. monocytogenes in the presenceof the IDR-1018 peptide (FIG. 10a , magnification×1000; FIG. 10b ,magnification×5000) at the concentration 50 μM, as described in Example4b.

FIG. 11 shows the number of colony-forming units measured followingplate seeding of samples of L. monocytogenes incubated for differenttime intervals, 1 h, 4 h, and 6 h, with a material functionalized withthe Bac-amp1 peptide (SOSF) or with the same material in the absence offunctionalization with the peptide (KSOS), as described in Example 5.

FIG. 12 shows a schematic view of the preparation of gold nanoparticlescovalently linked to peptide Bac-amp1 (Au—NPs-Bac-amp1), as described inExample 6.

FIG. 13 shows HPLC quantification of the peptide grafted to goldnanoparticles, as described in Example 8. Specifically the chromatogramsshow the absorbances obtained with a reference solution with initial (attime=0) peptide concentration (Bac-amp1), the supernatant solution atthe end of the functionalization reaction (unbound Bac-amp1), and thesupernatant solution after cleavage of the peptide from the surface ofthe nanoparticles (bound Bac-amp1).

FIG. 14 shows the titration curve for gold nanoparticlesfunctionalization with Bac-amp1 peptide as function of peptideconcentration. Immobilization yields obtained with Bac-amp1concentrations of 31, 75, 125 or 250 μM are shown.

FIG. 15 shows the bactericidal activity against Listeria monocytogenescells at the indicated concentrations (CFU/ml), expressed as percentageof bacterial cells killed, of gold nanoparticles either covalentlylinked to peptide Bac-amp1 (Au—NPs-Bac-amp1, grey bar) or without thepeptide (Au—NPs, black bar), at a concentration of 0.16 μM).

FIG. 16 shows the bactericidal activity against S. Typhimurium cells atthe indicated concentrations (CFU/ml), expressed as percentage ofbacterial cells killed, of the peptide Bac-amp1 covalently linked(concentration of 0.16 μM) to the gold nanoparticles (Au—NPs-Bac-amp1,grey bar) or gold nanoparticles without the peptide (Au—NPs, black bar).

FIG. 17 shows the long-term stability of bactericidal activity againstListeria monocytogenes for a period of up to 7 months.

FIG. 18 shows the bactericidal activity of the peptide free (at aconcentration of 0.16 μM) or covalently linked (at a concentration of0.16 μM) to nanoparticles against Listeria monocytogenes at theindicated concentrations (CFU/ml).

DEFINITIONS

The term “biofilm” according to the present invention refers tobacterial cells embedded within an extracellular matrix composed ofextracellular polymeric substances and attached to a living or abioticsurface.

The expressions “antibiofilm” or “biofilm inhibition activity” accordingto the present invention refers to the ability of peptides to prevent orinhibit the formation of biofilm of bacteria or to inhibit the growth ofbacteria in biofilm.

The expression “bactericidal activity” according to the presentinvention means the ability of peptides to kill planktonic (freeswimming) bacterial cells (bactericidal activity against the planktoniccells).

Potency of antibiofilm or bactericidal activity, as defined above, of apeptide compound (a protein chain consisting of at least 10 amino acids)against a specific microorganism is considered in the art to be suitablefor an use as antimicrobial or therapeutic agent when the minimuminhibitory concentration (MIC) and/or the minimum biofilm inhibitoryconcentration (MBIC) of that compound against that microorganism is nomore of 20 μM.

Active concentrations (MIC; MBIC) for such peptides higher than 20 μMwould result in uncompetitive costs for applications in differentindustrial fields.

The expression “antimicrobial activity” according to the presentinvention means the ability to prevent or eradicate a bacterialinfection in a living organism or on a product, such as a food, amaterial or an object.

The expressions “antimicrobial agent” or “antimicrobial composition”according to the present invention refers to a compound or acomposition, respectively, having an antimicrobial activity.

The expression “solvates” according to the present invention meanscomplexes of the peptides of the invention with the solvents in whichthe synthesis reaction takes place or in which they are precipitated orcrystallized. For example, a complex with water is known as a “hydrate”.

The expression “hybrid nanoparticle” according to the present inventionrefers to a nanoparticle composed of inorganic compounds, preferably oneor more metals, more preferably silver or gold and organic compounds,preferably one or more polymers, more preferably polyethylene glycol(PEG) made from polymerized ethylene glycol (ethane-1,2-diol) units. Thehybrid nanoparticles according to the invention comprise a polymercomponent with different functional end reactive groups, such as PEGdiamine and PEG-mercaptoethyl ether acetic or PEG diacid.

DETAILED DESCRIPTION OF THE INVENTION

As will be shown in the experimental section, the present inventors haveidentified that the peptides IDR-1018, having sequence VRLIVAVRIWRR (SEQID NO: 1) and Bac-amp1, having sequence VRLIVKVRIWRR (SEQ ID NO: 3) areendowed with a potent bactericidal activity showing MIC concentrationsin the low micromolar range against Listeria monocytogenes. This isunexpected in view of the activity of these peptides against othermicroorganisms disclosed in the prior art, which showed a selectiveantibiofilm activity and no bactericidal activity at the concentrationstested. Therefore, a first object of the invention is the use of atleast one peptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR, orVRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4), or a salt or solvate thereof, as an antimicrobial agentfor the prevention and/or treatment (removal) of contamination of aproduct or a surface by Listeria monocytogenes.

The above product may be any product susceptible of contamination byListeria, such as for example a food product, a material or an object.

Preferably, said product is an object, such as a container or a tool oroperating part of a machine, or a material, such as a packagingmaterial, preferably for the storage or processing of food.

Preferably, said surface is a surface a facility for the processing orstorage of food.

Preferably, the use according to the first object of the invention isfor the sanitization of facilities or machinery intended for processingfood products.

Said antimicrobial agent has a bactericidal activity against Listeriaplanktonic cells with a MIC at concentrations below 10 μM, preferablybelow 5 μM, more preferably below 1 μM.

Depending on the peptide and on the strain of Listeria involved in theinfection, the antimicrobial agent may also have an antibiofilmactivity.

Preferably, said antimicrobial agent has a combined bactericidalactivity and antibiofilm activity against Listeria.

Preferably, said at least one peptide is used at concentrations of up to10 μM, more preferably, below 8 μM, more preferably below 5 μM, evenmore preferably below 3 μM.

Preferably, said at least one peptide is used at concentrations between0.5 and 10 μM, more preferably between 0.5 and 8 μM, more preferablybetween 0.5 and 5 μM, even more preferably between 0.5 and 3 μM.

Particularly preferred is the use of the peptides according to theinvention VRLIVAVRIWRR or VRLIVKVRIWRR (SEQ ID NO:1, SEQ ID NO: 3), morepreferably, the use of a peptide VRLIVKVRIWRR, (SEQ ID NO: 3).

Salts or solvates suitable for the purposes of the invention are thosewhich do not lead to a conformational or stability modification of thepeptides according to the invention and, therefore, do not interferewith their biological activity.

As will be shown in the experimental section, the peptides of theinvention exhibit a strong bactericidal activity. The high bactericidalactivity of the peptides against Listeria monocytogenes allows obtainingan effective prevention or eradication of this microorganism withoutnecessarily having to associate it with other bactericidal compoundssuch as, for example, antibiotics.

Therefore, according to a preferred embodiment of the invention, said atleast one peptide is not in association with bactericidal compounds,preferably it is not in association with an antibiotic.

As shown in the experimental section, the peptides of the inventionexhibit a high bactericidal activity against Listeria monocytogenes and,in particular, also against strains of this bacterium isolated fromcontaminated food and environmental samples and belonging to serotype 4bor to serotype ½c, which are more virulent than the reference ATCC(American Type Culture Collection) or NTCC (National Type CultureCollection) strains commercially available, and are resistant to variousantibiotics (Ayaz et al, 2010, J. Food protection 73: 967-972).

Therefore, preferably said Listeria monocytogenes is anantibiotic-resistant Listeria monocytogenes serotype, more preferably itis a Listeria monocytogenes serotype resistant to one or moreantibiotics selected from cephalosporins, ampicillins, tetracyclines,erythromycins and penicillins. Preferably, said serotype is a serotype4b or ½c.

Surprisingly, the peptide having sequence VRLIVKVRIWRR (SEQ ID NO:3)shows a significantly more potent bactericidal activity against fieldstrains of this bacterium, in particular strains belonging to serotype4b or to serotype ½c, compared to that observed against the referenceATCC strain.

More preferably, said Listeria monocytogenes serotype is anantibiotic-resistant Listeria monocytogenes serotype, more preferably aListeria monocytogenes serotype resistant to one or more antibioticsselected from cephalosporins, ampicillins, tetracyclines, erythromycinsand penicillins, even more preferably a serotype 4b or ½c and said atleast one peptide is VRLIVKVRIWRR (SEQ ID NO:3).

According to a further preferred embodiment, the use according to thefirst object of the invention is as a preservative agent of foodproducts, in particular for the prevention and/or treatment ofcontamination thereof by Listeria monocytogenes.

Preferably, according to this embodiment, said use provides applying atleast one peptide according to the invention on the surface and/orinside the body of the food product. Alternatively, said use providesapplying at least one peptide according to the invention on the surfaceof food packaging.

As will be shown in the experimental section, the two peptides have ahigh stability to temperature and pH variations and are thereforeparticularly suitable to be used in the functionalization of materialsused for food packaging, maintaining their activity also following theproduction steps of the materials themselves.

In particular, the present inventors have found that the peptidesaccording to the invention show a high conformational stability even atextreme pH values (pH 1.0 or 11.0). In particular, the peptides of theinvention VRLIVKVRIWRR or VRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4),such as Bac-amp1, show a greater conformational stability at pH 11.0; onthe contrary, the peptides of the invention VRLIVAVRIWRR or VRLIVAVRIKRR(SEQ ID NO: 1, SEQ ID NO: 2), such as for example IDR-1018, show agreater conformational stability at pH 1.0.

Accordingly to the first object of the invention the peptide havingsequence VRLIVAVRIWRR (SEQ ID NO:1) is used for the packaging of foodproducts with a pH value in the range 1.0-4.0 preferably 4.0, while thepeptide having a sequence VRLIVKVRIWRR (SEQ ID NO:3) is used for thepackaging of food products having a pH up to 11, preferably of 8.0. Morepreferably, the use accordingly to the first object of the inventionprovides that in food packaging, a mixture is used of at least onepeptide having a sequence VRLIVAVRIWRR or VRLIVAVRIKRR (SEQ ID NO: 1,SEQ ID NO: 2) and at least one peptide having sequence VRLIVKVRIWRR orVRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4), preferably a mixture betweenthe peptide having sequence VRLIVAVRIWRR (SEQ ID NO: 1) and the peptidehaving sequence VRLIVKVRIWRR (SEQ ID NO: 3).

An association between the aforesaid peptides is particularlyadvantageous as it allows having an antimicrobial composition, which iseffective and resistant in a very wide pH range.

Preferably, in the use according to the first object of the invention,said at least one peptide is a mixture between at least one peptidehaving sequence VRLIVAVRIWRR or VRLIVAVRIKRR (SEQ ID NO: 1, SEQ ID NO:2) and at least one peptide having sequence VRLIVKVRIWRR or VRLIVKVRIKRR(SEQ ID NO: 3, SEQ ID NO: 4), preferably a mixture between the peptidehaving sequence VRLIVAVRIWRR (SEQ ID NO: 1) and the peptide havingsequence VRLIVKVRIWRR (SEQ ID NO: 3).

Furthermore, in accordance with the above, a second object of theinvention is an antimicrobial composition comprising a mixture of atleast one peptide having sequence VRLIVAVRIWRR or VRLIVAVRIKRR (SEQ IDNO: 1, SEQ ID NO: 2) and at least one peptide having sequenceVRLIVKVRIWRR or VRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4), preferablycomprising a mixture between the peptide having sequence VRLIVAVRIWRR(SEQ ID NO: 1) and the peptide having sequence VRLIVKVRIWRR (SEQ ID NO:3).

The peptide or mixture of peptides according to the invention is alsouseful in the treatment of diseases caused by Listeria monocytogenes, inparticular in the treatment of listeriosis.

Therefore, a third object of the invention is a peptide or a mixture ofpeptides having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR,or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4), or a pharmaceutically acceptable salt or solvate thereof, for use inthe treatment of an infection from Listeria monocytogenes in a subject,preferably for use in the treatment of listeriosis.

A fourth object of the invention is a method for the treatment of aninfection from Listeria monocytogenes in a subject, preferablylisteriosis, by administering a peptide or a mixture of peptides havingsequence VRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR(SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), or apharmaceutically acceptable salt or solvate thereof.

Preferably, according to the third or fourth object of the invention,said subject is a mammal, more preferably it is a human.

According to a preferred embodiment of the third or fourth object of theinvention, said peptide or mixture of peptide is or comprises a peptideVRLIVKVRIWRR (SEQ ID NO:3).

Salts or solvates according to the third or fourth object of theinvention are pharmaceutically acceptable salts which do not lead to aconformational or stability modification of the peptides according tothe invention.

Pharmaceutically acceptable acid addition salts include those formedwith hydrochloric, hydrobromic, acetic, phosphoric, lactic, pyruvic,acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic,glycolic, lactic, salicylic, oxaloacetic, methanesulfonic,ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic,naphthalene-2-sulphonic, benzenesulfonic and isetionic acids. Otheracids, such as oxalic acid, although not per se pharmaceuticallyacceptable, may be useful as intermediates for obtaining the compoundsof the invention and their pharmaceutically acceptable salts. Basesalts, considered acceptable, include ammonium salts, alkali metalsalts, for example potassium and sodium salts, alkaline earth metalsalts, for example calcium and magnesium salts and salts with organicbases, for example dicyclohexylamine and N-methyl-D-glucosamine.

According to a preferred embodiment of the third or fourth object of theinvention, said mixture of peptides is a mixture between at least onepeptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR (SEQ ID NO: 1, SEQID NO: 2) and at least one peptide having sequence VRLIVKVRIWRR, orVRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4), preferably it is a mixturebetween the peptide having sequence VRLIVAVRIWRR (SEQ ID NO: 1) and thepeptide having sequence VRLIVKVRIWRR (SEQ ID NO: 3).

In the case of oral administration, the association of the aforesaidpeptides allows overcoming the technical problem related to the decreasein the amount of peptide absorbed following the degradation of thepeptides to the acidic or basic pH encountered at the level of thegastrointestinal system. The compatibility of the two peptides at thetwo opposite ends of pH causes a greater amount of active peptide to beabsorbed and reach the site of action.

Preferably, said listeriosis is caused by an antibiotic-resistantListeria monocytogenes serotype, more preferably a serotype resistant toone or more antibiotics selected from cephalosporins, ampicillins,tetracyclines, erythromycin and penicillins. More preferably, saidListeriosis is caused by a strain belonging to serotype 4b or ½c.

More preferably, said Listeria monocytogenes serotype is anantibiotic-resistant Listeria monocytogenes serotype, more preferably aListeria monocytogenes serotype resistant to one or more antibioticsselected from cephalosporins, ampicillins, tetracyclines, erythromycinsand penicillins, even more preferably a serotype 4b or ½c and saidpeptide is or said mixture of peptides comprises VRLIVKVRIWRR (SEQ IDNO:3).

A fifth object of the invention is a pharmaceutical formulation orcomposition comprising at least one peptide having sequenceVRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4), or a pharmaceuticallyacceptable salt or solvate thereof, optionally in combination withpharmaceutically acceptable excipients or carriers.

According to a preferred embodiment of the fifth object of theinvention, said at least one peptide is a mixture between at least onepeptide having sequence VRLIVAVRIWRR or VRLIVAVRIKRR (SEQ ID NO: 1, SEQID NO: 2) and at least one peptide having sequence VRLIVKVRIWRR orVRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4), preferably a mixture betweenthe peptide having sequence VRLIVAVRIWRR (SEQ ID NO: 1) and the peptidehaving sequence VRLIVKVRIWRR (SEQ ID NO: 3).

The pharmaceutical formulations according to the invention comprisethose suitable for oral, parenteral (including subcutaneous,intradermal, intramuscular, intravenous and intra-articular) use, byinhalation (including powders of particularly small particles or mistswhich can be generated by means of various types of pressurized aerosoldispensers, nebulizers or insufflators), although the most suitableroute may depend, for example, on the recipient's condition.

The formulations can conveniently be presented in unit dosage form andcan be prepared by any of the well-known protocols of pharmaceuticaltechnique. All the methods provide for the step of associating theactive ingredient with the carrier, which constitutes one or more apharmaceutically acceptable accessory ingredients. In general, theformulations are prepared in a uniform manner by associating the activeingredient with finely divided solid or liquid supports or both andthen, if necessary, by formulating the product in the desiredformulation.

The formulations of the present invention suitable for oraladministration can be presented in discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; such as powder or granules; as a solution or suspension inan aqueous liquid or a non-aqueous liquid; or as a liquid oil-in-wateremulsion or as a liquid water-in-oil emulsion. The active ingredient mayalso be presented as bolus, electuary or paste. Several pharmaceuticallyacceptable carriers are described in standard formulation works, forexample Remington's Pharmaceutical Sciences by E. W. Martin. See alsoWang, Y. J. and Hanson, M. A., Journal of Parenteral Science andTechnology, technical report no. 10, Supp. Compositions for oraladministration include suspensions which may contain, for example,microcrystalline cellulose for imparting mass, alginic acid or sodiumalginate as suspending agent, methylcellulose as a viscosity enhancerand sweeteners or flavouring agents such as those already known in thepharmaceutical industry; immediate release tablets which may contain,for example, microcrystalline cellulose, calcium phosphate, starch,magnesium stearate and/or lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants such as those known inpharmaceutical techniques

Parenteral administration formulations include solutions for aqueous andnon-aqueous sterile injection that may contain antioxidants, buffers,bacteriostatic compounds, and solutes that make the formulationisotonic; aqueous and non-aqueous sterile suspensions, which may includesurfactants and thickeners. The formulations may be presented in unitdose containers or in multi-dose containers, such as vials and sealedvials, and may be stored in a lyophilized form requiring only theaddition of the sterile liquid support immediately prior to use.Compositions for administration of inhalation or nasal aerosols includesalt solutions which may contain, for example, benzyl alcohol or otherappropriate preservatives, which promote absorption to improvebioavailability and/or other solubilizing agents such as those known inthe pharmaceutical technique. In compositions for administration ofinhalation or nasal aerosols, the compound of the invention isadministered in the form of an aerosol from a pressurized container or anebulizer, using a suitable propellant, for exampledichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dose unit can be determined byproviding a valve to provide a measured amount. The preferred unitdosage formulations are those containing an effective dose, as describedabove, or an appropriate fraction, of the active ingredient.

It should be noted that, in addition to the ingredients specificallymentioned above, the formulations of the present invention may includeother conventional agents in use in pharmaceutical techniques takinginto account the type of formulation in question, for example thosesuitable for oral administration may include flavouring agents. Thecompounds of the invention are also appropriately administered ascontinuous and controlled release systems. Suitable examples ofcontinuous release systems of the invention include suitable polymericmaterials, such as semi-permeable polymeric matrices, such as films ormicrocapsules; specific hydrophobic materials, such as emulsions insuitable oils or ion exchange resins; and derivatives of the compound ofthe invention, such as, for example, a soluble salt. According toembodiments of the use according to the first object of the invention,also in combination with other preferred embodiments described above,the at least one peptide may be applied to the surface of the product orsurface to be protected or treated from contamination by Listeria bymeans of incorporation in a coating applied on said surface by covalentbonding to reactive groups present on said surface.

This allows to prolong the period of permanence of bactericidalconcentrations of the peptide on the surface to be protected fromcontamination and thus to exert a long lasting protective activity onsaid surface.

According to one embodiment of the use according to the first object ofthe invention, said at least one peptide is applied as a coatingcomposition on the surface of said product or on said surface.

In this case, the use of the peptide or mixture of peptides according ofthe first object of the invention provides applying a liquidantimicrobial composition comprising said at least one peptide and,optionally, film forming polymers, to the surface of said product andthen drying, thereby forming a coating.

A sixth object of the invention is therefore a coating compositioncomprising at least one peptide having sequence VRLIVAVRIWRR, orVRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: and SEQ ID NO: 4).

Said coating composition preferably further comprises film-formingagents. These are compounds that are able to form a film on the surfaceof said object or material, which favours the permanence of the peptideor mixture of peptides thereon. Preferably, said film forming agent is afilm-forming polymer.

A seventh object of the present invention is a product having at leastone surface covered with a coating adherent to said surface, saidcoating comprising at least one peptide having sequence VRLIVAVRIWRR, orVRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: and SEQ ID NO: 4) and, optionally, a film forming polymer.

Said coating may cover a part or the whole of the said surface.

Preferably, said product is a material or an object, as above defined.

According to a preferred embodiment of the sixth or seventh object ofthe invention, said at least one peptide is a mixture between at leastone peptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR (SEQ ID NO: 1,SEQ ID NO: 2) and at least one peptide having sequence VRLIVKVRIWRR, orVRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4), preferably a mixture betweenthe peptide having sequence VRLIVAVRIWRR (SEQ ID NO: 1) and the peptidehaving sequence VRLIVKVRIWRR (SEQ ID NO: 3).

According to another embodiment of the use according to the first objectof the invention, said at least one peptide is linked with a covalentbond to the surface of said product or on said surface.

In fact, as will be shown in the Examples, the present inventors havefound that when the N-terminal group of the above peptide is linkedcovalently to a surface, the bactericidal activity is maintainednotwithstanding the conformational constrains of the linked peptide.

In this case, the use of the peptide or mixture of peptides providesthat the peptide or mixture of peptides according to the invention isapplied by forming a covalent bond with reactive groups present on asurface. This allows obtaining a product with a surface characterized bya bactericidal activity against Listeria monocytogenes, which is stablefor long periods of time.

According to another embodiment of the use according to the first, thirdof fourth object of the invention, said at least one peptide may belinked with a covalent bond to the surface of nanoparticles that can beused as antimicrobial agents.

Accordingly, an eighth object of the invention is a product having atleast one surface covalently linked to at least one peptide havingsequence VRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR(SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: and SEQ ID NO: 4).

Preferably, said peptide is linked to said surface by means of acovalent bond between the N-terminal amino group of the peptide and achemical group on said surface.

Said peptide may be linked to a part or the whole of the said surface.Preferably, according to the eight object of the invention, said atleast one peptide is a mixture between at least one peptide havingsequence VRLIVAVRIWRR, or VRLIVAVRIKRR (SEQ ID NO: 1, SEQ ID NO: 2) andat least one peptide having sequence VRLIVKVRIWRR, or VRLIVKVRIKRR (SEQID NO: 3, SEQ ID NO: 4), preferably a mixture between the peptide havingsequence VRLIVAVRIWRR (SEQ ID NO: 1) and the peptide having sequenceVRLIVKVRIWRR (SEQ ID NO: 3). Said product may be any product having asurface bearing chemical groups able to form a covalent bond with theN-terminal amino group of the peptide. Preferably said chemical groupsare selected from a carboxyl groups, excited hydroxyl radicals,activated alkoxy groups or activated aldehyde or ketone groups.

Preferably, said product having at least one surface is an object, suchas for example a container for storing food, a tool or operating part ofa machine, for example used in food processing or a material, such asfor example a packaging material, a polymer, a metal or a semiconductor.

Techniques for binding amino groups of peptides or proteins to surfacesare known to those skilled in the art and vary according to the materialused.

For example, in the case of materials having surfaces in metal (gold,platinum silver) and semiconductors (titanium, zinc, tin, zirconium, andgermanium), a silanization process may be used. This involves, forexample, the reaction with the material to be treated of a mixture ofsulfuric acid (H₂SO₄) and oxygen peroxide (H₂O₂), which are capable ofactivating the aforementioned surfaces by creating bonds of surfaceatoms and hydroxyl groups (—OH) easily replaceable by more stable bondssuch as Si—C or Au—S. The activated surfaces can covalently bind thepeptides of interest following treatment with a silanizing agent, suchas aminopropyldimethylethoxysilane or aminopropyltritoxysilane, and acompound having two functional groups capable of forming the peptidecovalent bond with the peptide amino groups, such as gluteraldehyde orbis-succininimide. These treatments are typical of the chemistry ofaqueous solutions and for this reason they are called wet processes,which are advantageous because they do not require particulartechnological equipment but are limited to preferably rigid materialsthat can be wetted and dried without difficulty.

In the case of plastic or polymeric surfaces, these can instead befunctionalized to hook the peptides of interest either by applying thewet processes described above, or by applying high electromagneticenergy radiation (for example by laser, ultraviolet radiation, gammarays). For so-called soft materials such as non-rigid plastics, wetprocesses may not be completely effective, so the so-called dryfunctionalization processes based on the interaction of the surface witha gaseous plasma or an electromagnetic radiation are preferred. Theinteraction of the surface of a polymer with electromagnetic radiationcauses surface activation through the breaking of accessible polymericbonds, so the C═C bonds become —C—C— allowing the subsequent chemicalmodification of the surface itself. The same operating principle appliesto the other method of activation of the polymer surfaces and consistsin the treatment thereof with ionized gas (gaseous plasma). This processis particularly advantageous because, since plasma is cold, thetemperature of the treated material does not reach high values withrespect to the ambient temperature. This method requires low pressure(0.1-100 Pa) and the presence of a working gas (usually N2, O2 or Ar,CF4) [Hegemann, Dirk, Herwig Brunner, and Christian Oehr. “Plasmatreatment of polymers for surface and adhesion improvement.” Nuclearinstruments and methods in physics research section B: Beam interactionswith materials and atoms 208 (2003): 281-286].

According to a particularly embodiment of the eighth object of theinvention, said product consists in nanoparticles. As will be shown inthe experimental section, when the peptide of the invention is linked onthe surface of nanoparticles, the bactericidal activity is surprisinglysignificantly increased. Furthermore, the peptide shows stability over along period of time.

Accordingly, a particularly preferred object of the invention arenanoparticles comprising, covalently linked to their surface, at leastone peptide according to the invention as described above, havingsequence VRLIVAVRIWRR, or VRLIVAVRIKRR, or VRLIVKVRIWRR, or VRLIVKVRIKRR(SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4).

Preferably, said at least one peptide is a mixture of at least onepeptide having sequence VRLIVAVRIWRR, or VRLIVAVRIKRR (SEQ ID NO: 1, SEQID NO: 2) and at least one peptide having sequence VRLIVKVRIWRR, orVRLIVKVRIKRR (SEQ ID NO: 3, SEQ ID NO: 4), preferably a mixture betweenthe peptide having sequence VRLIVAVRIWRR (SEQ ID NO: 1) and the peptidehaving sequence VRLIVKVRIWRR (SEQ ID NO: 3).

Preferably, said peptide is linked to said surface of the nanoparticlesby means of a covalent bond between the N-terminal amino group of thepeptide and a chemical group on said surface.

Preferably, the N-terminal amino group of said at least one peptide iscovalently bound to a chemical group on the surface of saidnanoparticles selected from carboxyl groups, excited hydroxyl radicals,hydroxyl radicals, activated alkoxy groups or activated aldehyde orketone groups.

Preferably, said at least one peptide is linked substantially uniformlyover the whole surface of the nanoparticles.

Preferably, said nanoparticles have an average hydrodynamic diametermeasured by dynamic light scattering (DLS) comprised between 5 and 90nm, preferably between 5 and 70 nm, preferably between 5 and 50 nm,preferably between 5 and 40 nm, preferably between 5 and 30 nm,preferably between 5 and 30 nm, or more preferably of 15 nm.

Preferably, said nanoparticles are hybrid nanoparticles, more preferablyare hybrid metal nanoparticles, even more preferably silver or goldhybrid nanoparticles.

Preferably, said hybrid nanoparticles contain a polymer as organiccomponent, preferably selected from PEG molecules functionalized withdifferent reactive groups, such as PEG diamine and PEG-mercaptoethylether acetic and PEG diacid.

The above nanoparticles may be used to deliver the peptides according tothe invention to their site of activity. Accordingly, in a preferredembodiment of the first, third or fourth object of the invention said atleast one peptide is linked to the surface of nanoparticles according tothe invention as above described.

Nanoparticles particularly suitable for therapeutic delivery of activecompounds are natural materials or derivatives (chitosan, dextrane,gelatine, liposomes, alginates, starch), dendrimers, fullerenes, polymercarriers (polylactic acid, poly(cyano) acrylates, polyethileinemine,polycaprolactone), metallic nanoparticles, quantum dots, silicananoparticles. Accordingly, in the above embodiment of the tenth,eleventh or twelfth object of the invention, the nanoparticles are madeof a material or a combination of material selected from the above.

As will be shown in the experimental section, the present inventors havesurprisingly found that the nanoparticles according to the invention areable to increase the bactericidal activity of the peptides not onlyagainst Listeria but also against bacteria different from Listeriamonocytogenes.

Therefore, the above described nanoparticles may be advantageously usedto prevent or treat contamination or infections by many other bacteria.

The above nanoparticles according to the invention can be dispersed in aliquid composition that can be used as an antimicrobial composition.

Accordingly, a ninth object of the invention is a liquid antimicrobialcomposition comprising the nanoparticles according to the invention, asdescribed above.

A tenth object of the invention is the use of the above nanoparticlesaccording to the invention as an antimicrobial agent for the preventionor treatment of contamination of a product or surface by bacteria.Preferably, said bacteria are selected from Listeria monocytogenes andS. typhimurium.

Preferably, the use according to the ninth object of the invention isfor the sanitization of facilities or machinery intended for processingfood products.

According to a further preferred embodiment, the use according to theninth object of the invention is as a preservative agent of foodproducts, in particular for the prevention and/or treatment ofcontamination thereof by Listeria monocytogenes, in particular for theprevention and/or elimination of bacterial contamination thereof and ofthe food contained therein.

Preferably, according to a ninth embodiment, said use provides applyingat least one peptide according to the invention on the surface and/orinside the body of the food products. Alternatively, said use providesapplying at least one peptide according to the invention on the surfaceof food packaging material.

The nanoparticles are also suitable for the delivery of the peptides toa patient.

Accordingly, a tenth object of the invention are nanoparticles, asdescribed above, for use in the treatment of a bacterial infection in asubject, preferably for use in the treatment of an infection againstListeria monocytogenes. An eleventh object of the invention is a methodfor the treatment of a bacterial infection in a subject, byadministering nanoparticles, as described above. Preferably, accordingto the tenth and eleventh object of the invention, said subject is amammal, more preferably it is a human.

A twelfth object of the invention is a pharmaceutical compositioncomprising nanoparticles as described above, optionally in combinationwith pharmaceutically acceptable excipients or carriers.

Preferably, in the above embodiment of the tenth, eleventh or twelfthobject of the invention, the nanoparticles are: natural materials orderivatives (chitosan, dextrane, gelatine, liposomes, alginates,starch), polymer carriers (polylactic acid, poly(cyano) acrylates,polyethileinemine, polycaprolactone), metallic nanoparticles, silicananoparticles.

EXAMPLES Example 1—Synthesis of Peptides

The Bac-amp1 and IDR-1018 peptides, having the sequences shown in table1, were synthesized by solid phase peptide synthesis using theFluoromethoxycarbonyl (Fmoc) protecting group.

TABLE 1 Peptide Sequence Bac-amp1 VRLIVKVRIWRR-NH₂ (SEQ ID NO: 3)IDR-1018 VRLIVAKVRIWRR-NH₂ (SEQ ID NO: 1)

The Rink-Amide MBHA resin with a degree of substitution of 0.5 mmol/gwas used as a solid support. The resin is provided with a “linker”,which provides an amide bond and releases the amided peptide to theC-terminus.

At the end of the synthesis, the protecting group was removed bytreatment with a solution of 40% (v/v) piperidine in DMF while thedetachment from the resin and the removal of the protecting groups onthe side chains of the amino acids was obtained by treatment with anacid solution composed of 95% trifluoroacetic acid, 2.5%triisopropylsilane and 2.5% H2O (v/v/v).

After detachment from the solid support, each peptide was precipitatedin cold ethyl ether, at −20° C. To recover the precipitate, each samplewas centrifuged at 3500 rpm for 5 minutes. The precipitate was dissolvedin a mixture of CH3CN/H2O (95:5), frozen and lyophilized.

Example 2—Analysis of Peptide Stability

In a first set of experiments, the stability of Bac-amp1 and IDR-1018 atdifferent temperatures and pH conditions was assessed.

The stability of the Bac-amp1 and IDR-1018 peptides was assessed bystudying their secondary structure with Circular Dichroism (CD)spectroscopy in the presence of Sodium Dodecyl Sulfate (SDS) micelles ata concentration of 10 mM to mimic the condition of the cell membrane, indifferent temperature and pH conditions.

CD assays were performed with a Jasco J-810 Spectropolarimeter equippedwith a thermostatted cuvette compartment. The samples were loaded into a0.1 cm-long quartz cuvette (Hellma Analytics) and the spectrum wasanalyzed in the 190 nm to 250 nm range at temperatures of 15° C. or 90°C. for each sample.

The CD spectra were acquired at a scanning speed of 20 nm/min, and therecorded results represent an average of 5 scans. The spectra at 90° C.were collected at time zero and after 2 hours to evaluate thethermostability of peptides at high temperatures.

The average ellipticity of the residues ([θ], degrees cm² dmol⁻¹) wascalculated using the following equation:[θ]=100θ/cn1where θ represents ellipticity (m degrees), c is the concentration (mM)of the peptide, n is the number of residues. The secondary structure wascalculated through the DICHROWEB site (Whitmore L and Wallace B A, 2004,Nucleic Acids Research 32: 668-673, Whitmore L and Wallace B A, 2008,Biopolymers 89:392-400, Lobley et al, 2002, Bioinformatics 18:211-212),using three different algorithms (SELCON3, CONTIN-LLe CDSSTR) (SreeremaN and Woody R W, 1993, Analytical Biochemistry 287:252-260; Sreerema Net al, 1999, Protein Science 8:370-380, Provencher S W and Glockner J,1981, Biochemistry 20:33-37, Van Stokkum I H M et al, 1990, AnalyticalBiochemistry 191:110-118, Compton L A and Johnson W C, 1986, AnalyticalBiochemistry 155:155-167, Manavalan P and Johnson W C, 1987, AnalyticalBiochemistry 167:76-85, Sreerama N et al, 2000, Protein Science8:370-380) and selecting as a comparison data set the SMP180 protein,which includes a high number of soluble and membrane proteins(Abdul-Gader A et al, 2011, Bioinformatics 27:1630-1636).

Example 2a—Thermostability Assay

0.1 g/L of Bac-amp1 or IDR-1018 peptide were dissolved in 100 mM sodiumacetate buffer at pH 6 and 10 mM SDS. The prepared samples wereincubated for two hours at 15° C. or 90° C. A CD analysis was performedon samples stored at 15° C. and before and after incubation for twohours at 90° C. The results obtained for Bac-amp1 or IDR-1018 are shownin FIGS. 1 and 2, respectively.

As shown in FIG. 1, all Bac-amp1 CD spectra have two negative bands at215 nm and 222 nm which indicate a conformational state of the peptideconsisting of alpha-helix structures. Furthermore, no significantchanges were found in the sample spectra at 90° C., even after 2 hoursincubation at this temperature. These data demonstrate that Bac-amp1maintains its structure unchanged in the range from 15 to 90° C. and istherefore characterized by a high thermostability.

As shown in FIG. 2, all IDR-1018 CD spectra have a single negative bandat about 212 nm which indicates a conformational state of the peptideconsisting of mainly beta-sheet structures. Furthermore, also with thispeptide, no significant changes were found in the sample spectra at 90°C., even after 2 hours incubation at this temperature. Therefore, alsothe IDR-1018 peptide is characterized by high thermostability. Thedifferent conformation of the two peptides accounts for the differentbactericidal activity towards specific microorganisms, discussed below,namely the ability to contrast with different effectiveness the growthof pathogenic bacteria such as Listeria monocytogenes.

Example 2b—pH Stability Assay

0.1 g/L of Bac-amp1 or IDR-1018 peptide were dissolved in differentsolutions at 100 mM concentration, in particular: potassium chloride-HCL(pH 1); glycine-HCL buffer (pH 2); sodium acetate buffer (pH 4 and 6);tris-HCL buffer (pH 8); glycine-NaOH buffer (pH 10);sodium-bicarbonate-NaOH (pH 11). After an hour of incubation at 25° C.,samples of each solution containing SDS at a concentration of 10 mM wereprepared. The resulting samples and a glycine-HCL buffer sample withoutaddition of SDS were incubated at 37° C. for another 24 hours andanalyzed by CD. The results obtained are shown in FIGS. 3 and 4.

In both figures, it is seen that, for both peptides, the samples at pH 2without addition of SDS show a negative band shifted towards 198 nmwhich indicates the presence of an unordered “random coil” structure,while all the other samples show an ordered structure, of alpha-helixtype for Bac-amp1 and of beta-sheet type for IDR-1018.

As shown in FIG. 3, all Bac-amp1 CD spectra recorded at different pHconditions in the presence of SDS indicate that the peptide takesalpha-helix conformations, in all the analyzed conditions, to which thepeptide functionality is associated.

As shown in FIG. 4, the CD spectrum of IDR-1018 recorded at pH 2 withoutthe addition of SDS has a negative band shifted towards 198 nm, whichindicates that the peptide under these conditions takes a differentconformational state; the CD spectra of this peptide recorded in thepresence of SDS at different pH conditions indicate that the peptidetakes beta-sheet conformations.

Overall, these results show that the Bac-amp1 peptide maintains itsstructural integrity over the entire pH range better than the IDR-1018peptide. In fact, the analysis of the CD spectra of the IDR-1018 peptidein comparison with Bac-amp1 clearly indicates an increase in thepercentage of unordered random coil structure in the pH range of8.0-11.0 for IDR-1018 and in the range of 1.0-4.0 for Bac-amp1.Therefore, the two peptides exhibit a different conformational stabilityin specific pH ranges and consequently a different functionality, interms of bactericidal activity since it is strictly dependent on theirconformation, whether alpha-helix or beta-sheet.

In the light of these results, the present inventors have shown that thecombination of the two peptides ensures bactericidal activity in a widepH range, which is a feature that finds useful application in the foodindustry.

Example 2c—Stability Analysis Under Use Conditions

Samples of mozzarella brine were incubated for 24 hours at 4° C. in thepresence of Bac-amp1 or IDR-1018 or in their absence (control). Thebrine was obtained from skimmed, pasteurized kneading water, added with3-5% NaCl and acidified with lactic or citric acid up to pH 3.8-3.9. Thethree samples were analyzed by HPLC on a mBondpack C18 reverse phasecolumn (RP).

In FIG. 5, a chromatogram obtained for each sample tested is shown; asshown by this chromatogram, neither precipitation nor degradation of thepeptides occurred during the incubations, which therefore resultedcompletely stable in the brine.

Example 3—Bactericidal Activity Assay

The bactericidal activity of the Bac-amp1 and IDR-1018 peptides wasevaluated against both Gram+ (Listeria monocytogenes and Staphylococcusaureus) and Gram− (Salmonella typhimurium) pathogenic bacteria.

For the three selected bacterial species, certified and characterizedstrains were used, indicated in Table 2.

The evaluation of the minimum bactericidal concentration (MBC) wascarried out as described in Bilikova et al, (2015, Peptides 68:190-196)and the evaluation of the effective concentration to reduce 50% of thebacterial population (EC50) of the two peptides was carried out with themicro-dilutions of the broth as described in Wang H X and Ng T B (2003,Peptides 24:969-972).

Standard deviations for triple incubations of each plate and EC50evaluation were determined using GraphPad Prism version 6.00 (Graph-PadSoftware, La Jolla Calif. USA)

Listeria monocytogenes

As regards Listeria monocytogenes, five field strains were added to thetrial (wild strains) SS1 (serotype 4b), SS2 (serotype 4b), SS3 (serotype4b), SS4 (serotype 4b), SS5 (serotype ½c) shown in Table 2. Thesestrains were isolated from 200 samples of food waste, in particular fishproducts and milk derivatives, obtained from workbenches of foodcompanies. All samples were collected in a context of official controls.Isolation of Listeria monocytogenes strains was performed according tothe ISO-11290-1 standard. In brief, 25 grams of sample were homogenized(1:10 w/v) in Half Fraser Broth culture medium (AES Laboratoire, Routede Dol, Combourg, France) and incubated at 37° C. for 18 hours. 1 ml ofculture broth was transferred to Fraser Broth culture medium (Fraser J Aand Sperber W H, 1988, J Food Protect 51(10):762-765) and incubated at37° C. for 24 hours. Subsequently, the enriched was streaked on ALOA andOxford agar (Oxoid, Basingstoke, UK) at 37° C. for 24 hours. Listeriamonocytogenes colonies were identified biochemically with Listeria API(BioMérieux, Marcy l'Etoile, France).

For all five field strains obtained, serotyping was performed usingprotocols which are certified (Pulsed Field Gel Electrophoresis PFGE,PulseNet using the Ascl and Apal restriction enzymes) and reported inthe Analytical Bacteriology Manual of Food and Drug Administration (FDA)of the United States and through the use of commercial antibodies forsomatic (0) and flagellar (H) antigens (Denkan Seiken Co. Ltd, Tokyo,Japan). In particular, the SS1-SS4 strains were found to belong toserotype 4b, which is the most pathogenic and aggressive for humans,responsible for at least 50% of listeriosis cases, while the SS5 strainbelongs to serotype ½c. A control stock suspension was prepared in which10³ CFU of L. monocytogenes (ATCC strain and SS1, SS2, SS3, SS4 and SS5strains isolated as described above) were inoculated in 10 ml of HalfFraser Broth and serial dilutions (100 to 0.01 μM) of the suspensionwere performed. 5 mM stock solutions of the two peptides in DMSO werethen prepared and serial dilutions (100 to 0.01 μM) were performed inFraser Broth, which were inoculated with 10³ CFU (colony forming units)of L. monocytogenes and incubated for 6 hours at 37° C. At the sametime, control samples were treated in the same way but without theaddition of the two peptides. 50 μl of each bacterial suspension wasseeded in different culture plates: blood agar and ALOA (Oxoid,Basingstoke, UK), which were then incubated for 24-48 hours at 37° C.Each dilution series included control plates with bacteria and DMSOalone without the peptide. The assay was performed in parallel with anATCC strain and with 2 wild strains isolated from sea foods as describedabove.

Salmonella typhimurium

A control stock suspension was prepared in which 10³ CFU of S.typhimurium (ATCC13311 strain) were inoculated in 10 ml of BPW (Oxoid,Basingstoke, UK). Then, 5 mM stock solutions of the two peptides wereprepared and serial dilutions (100 to 1 μM) were performed in BPW,inoculated with 10³ CFU of S. typhimurium and incubated for 6 hours at37° C. 50 μl of each bacterial suspension was seeded in petri disheswith blood agar or chromogenic agar (Oxoid, Basingstoke, UK) andincubated for 20 hours at 37° C. Each dilution series included controlplates inoculated with DMSO without the peptide and control plates withbacteria alone.

Staphylococcus aureus

A control stock suspension was prepared in which 10³ CFU of S.typhimurium (6571 strain) were inoculated in 10 ml of BPW. Then, 5 mMstock solutions of the two peptides were prepared and serial dilutions(100 to 1 μM) were performed in BPW, which were inoculated with 10³ CFUof S. aureus and incubated for 6 hours at 37° C. Also in this case, thedilution series included control plates inoculated with DMSO without thepeptide and control plates with bacteria only.

50 μl of each bacterial suspension were poured onto petri dishes withblood agar or rabbit plasma fibrinogen agar and incubated for 20 hoursat 37° C.

In all the investigated experimental conditions, the plate countingmethod was used to estimate the bactericidal activity of the peptides.Specifically, the number of colonies grown on plates with agar seededwith bacterial suspensions in the absence or in the presence ofindividual dilutions of peptides was counted and compared. The standarddeviations of the triple incubations of each plate were determined usingstatistical software.

All bactericidal activity assays were performed using 2 log CFUs (whennot otherwise specified), which represents a realistic approximation ofthe levels of contamination that may be contained in fresh foodproducts. FIG. 6 shows the dose-response curve obtained with theBac-amp-1 peptide against S. aureus (NCTC), S. thyphimurium (ATCC), L.monocytogenes (ATCC), L. monocytogenes (SS1) and L. monocytogenes (SS2).

Based on the dose-response curves obtained with both peptides, thevalues of EC50 (effective peptide concentration to reduce 50% of thebacterial population present in the medium) against the commerciallyavailable bacterial strains (ATCC, NTCC) and Listeria field strains(SS1-SS5), for the other three Listeria strains isolated from food andwork surfaces, the available data allow identifying only theconcentration threshold values at which the two peptides are stillactive (Table 2).

TABLE 2 Pathogenic species EC50 IDR-1018 EC50 Bac-amp1 S. aureus (NCTC)1.58 μM 1.66 μM S. thyphimurium (ATCC) 3.12 μM 2.83 μM L. monocytogenes(ATCC) 0.22 μM 0.55 μM L. monocytogenes (SS1) 0.69 μM 0.28 μM L.monocytogenes (SS2) 0.40 μM 0.08 μM L. monocytogenes (SS3) <25 μM <1 μML. monocytogenes (SS4) <25 μM <1 μM L. monocytogenes (SS5) <25 μM <1 μM

The data reported in Table 2 show that for both peptides there is apowerful bactericidal activity against L. monocytogenes compared to thatrecorded against S. aureus and S. thyphimurium strains, which is ofmoderate magnitude.

In fact, both peptides show an EC50 value of less than 0.7 μM on allListeria strains. Furthermore, while the IDR-1018 peptide shows a morepotent activity against the commercial Listeria strains, the Bac-amp1peptide seems to have a particular action specificity towards the 5field strains, with values of EC50 included between <1 μM and 0.28 μM.These results suggest that in the case of Bac-amp1 there is a precisemechanism of antimicrobial action against Listeria which involvestranslocation of the bacterium through the membrane to interact andinhibit specific intracellular targets involved in vital metabolicprocesses.

This activity of the Bac-amp1 peptide represents a very relevant datumfrom an applicative point of view if we consider that generally,microbial strains isolated from the environment and/or food showadaptation or resistance to antibiotics and disinfectants, probably dueto the presence of mobile genetic elements carrying resistance genes oraltered permeability of the bacterial cell wall.

Example 4—Antibiofilm Activity Assay

The antibiofilm activity of the Bac-amp1 and IDR-1018 peptides wastested by analyzing their ability to inhibit the formation of bacterialbiofilms of L. monocytogenes.

Since it is known that the antibiotic activity of antimicrobial agentsis strongly dependent on the experimental approach, which could favourthe inhibition of biofilms and overestimate their real effectiveness, inthe present study the assessment of the ability of such peptides tocounteract the formation of bacterial biofilms was carried out on steeldiscs, an inert material widely used in the food industry.

Example 4a—Antibiofilm Activity Assay with Crystal Violet Staining

The ability of peptides to prevent the formation of L. monocytogenesbiofilms was determined according to the microplate assay described inG. Di Bonaventura et al. (2008, J Appl Microbiol 104(6):1552-61) withsome modifications.

L. monocytogenes cultures (ATCC7644 and EURL12MOB098LM) were prepared byinoculating Brain Heart Infusion Broth broth (Sigma-Aldrich, City andState) at 37° C. up to a logarithmic growth phase. 10 ml of bacterialsuspension were then centrifuged the cell pellet was washed in PBS anddiluted in BHI broth, reaching the concentration of about 10³ CFU/ml ofstandardized inoculum.

The biofilm formation assays were conducted using as target surfaces theAisi 304 stainless steel discs having a diameter of 14.5 mm (SS) used inthe food industry as food contact surfaces. The discs were placed on24-well Falcon® tissue culture plates, (Thermo Fisher Scientific Inc.,Waltham, Mass., USA), with flat bottom and lid. Before use, thestainless steel discs were immersed in 10% acetone and left under weakstirring at room temperature for 30 min. After washing in ultrapuresterile water, the discs were incubated in ethanol 99.8% for 10 minutesunder weak stirring and then washed in ultrapure sterile water, dried,packaged and sterilized at 121° C., 1 atm for 15 minutes.

In each set of experiments, 600 μl of standardized inoculum in thepresence or absence of each of the peptides at the concentration of 12.5μM, 25 μM or 50 μM were added to 24-well culture plates containing thepreviously treated stainless steel materials. BHI Broth was used as anegative control. The plates were incubated at 37° C. for 72 hours understatic conditions. The cell count of L. monocytogenes, according to theISO 11290-2:98 method. After incubation, the SS discs were washed threetimes with PBS and placed in a new plate to dry at 60° C. for 1 hour. 1ml of 0.2% crystal violet in 95% ethanol was added to each well to stainthe bacterial cells adhered to the surface of the SS discs. After weakstirring for 15 minutes, the SS discs were washed three times withsterile water and then transferred to a new plate to dry at 37° C. for 1hour. Quantitative analysis of biofilm production was performed byadding 1 ml of 33% acetic acid, which removes the dye from the adheredcells. 200 μl of the obtained solution were transferred to a microplateand the crystal violet level (OD) was measured at 492 nm.

The amount of bacterial biomass for each incubation condition wasrepresented by “box plots” diagrams; the points outside the box wereconsidered as anomalous values (outliers). Statistical significancetests were performed by applying non-parametric variance analysis(Kruskal-Wallis test) followed by multiple pairs comparisons usingDunn's test with Bonferroni correction (p<0.05). Statistical assays wereperformed using MICROSOFT® EXCEL 2000/XLSTAT©-PRO, a tool forstatistical analysis in the spreadsheet programme developed byMicrosoft.

As shown in FIG. 7, the Bac-amp1 peptide showed a significant antibioticactivity with a reduction of 70%-80% microbial biomass at concentrationsof 20-25 μM and 100% at concentrations of 25-50 μM corresponding to thevalue of MBIC₁₀₀ (minimum biofilm inhibiting concentration).

As shown in FIG. 8, the IDR-1018 peptide, as opposed to Bac-amp1, doesnot 100% inhibit the formation of bacterial biofilm in any of theexperimental conditions adopted, reaching a maximum inhibition of 81.5%at the 50 μM concentration.

These results show that the Bac-amp1 peptide is capable pf totallyinhibiting the formation of antibiofilm by Listeria, while IDR-1018 hasa weaker antibiofilm activity.

These data also suggest that the conformation adopted by the Bac-amp1peptide is more suitable than that of the IDR-1018 peptide in preventingthe growth of the biofilm produced by Listeria monocytogenes.

Example 4b—Antibiofilm Activity Assay with SEM

The activity of the Bac-amp1 and IDR-1018 peptides on L. monocytogenesbiofilms was further investigated by scanning electron microscopy (SEM).

Specifically, in this example the three-dimensional biofilmarchitecture, developed on stainless steel materials, was studied.

The Aisi 304 stainless steel discs with a diameter of 14.5 mm (SS) weretreated with 600 μl of standardized inoculum in the presence or absenceof each of the peptides at the concentration of 25 μM or 50 μM, or withcontrol medium, according to the same method of Example 4a andrepeatedly washed in sterile PBS to remove planktonic cells and fixatedon a new plate with 1 ml of Karnovsky fixative containing 5%glutaraldehyde, 4% paraformaldehyde in buffer 0.064 M (ElectronicMicroscopy Science, Hatfield, Pa., USA) for 1 hour at 4° C. Thereafter,the samples were washed in 0.1 M cacodylate sodium buffer at pH 7.4(Electronic Microscopy Science, Hatfield, Pa., USA) and the fixatedcells were dehydrated in a series of aqueous acetone solutions atdifferent concentrations, in particular 25%, 50%, 75% and 100%. Thesamples were glued onto polished aluminium sample holders (ElectronicMicroscopy Science, Hatfield, Pa., USA) and uniformly vacuum coated witha 20-nm thick gold layer with the K950 high-vacuum Turbo Evaporator anda K 350 coupling (Emitech Ltd, Ashford, UK). The coated samples wereexamined at the JEOL 6700 FEG Field Emission Electron Microscope (SEMJeol, City and State) with magnification from 1000× to 5000×. For eachsample, a number of images (5-10), acquired as electronic images wererandomly analyzed.

It was observed that the treatment with Bac-amp1 significantly reducesthe formation of biofilm already at 25 μM while the best result isobserved at 50 μM.

FIG. 9 shows microscopic images showing the effect of the Bac-amp1peptide at a concentration of 50 μM on biofilm formation.

As seen in FIG. 9a , the 1000× image of the samples treated withBac-amp1 shows cells that are more rare and scattered than those ofuntreated control samples, whose image is shown in FIG. 9 b.

As shown in FIG. 9c , the 5000× image of the samples treated withBac-amp1 shows the near absence of cells, which instead are present inthe untreated control samples, whose image is shown in FIG. 9 d.

FIG. 10 shows microscopic images showing the effect of the IDR-1018peptide at a concentration of 50 μM on biofilm formation.

As seen in FIG. 10a , the 1000× image of the samples treated withIDR-1018 shows a greater number of cells compared to the 1000× image ofthe samples treated with Bac-amp1, shown in FIG. 9a . Furthermore, ascan be seen in FIG. 10b , the 5000× image of the samples treated withIDR-1018 shows a greater number of cells compared to the 5000× image ofthe samples treated with Bac-amp1, shown in FIG. 9 c.

These results confirm that the Bac-amp1 peptide is capable of exertingits bactericidal activity also on biofilms and has a capacity to inhibitthe formation of biofilm produced by L. monocytogenes that is clearlysuperior to that of the IDR-1018 peptide.

Example 5—Bactericidal Activity Assay on Supports Functionalized withBac-Amp1

A solid support consisting predominantly of silicon oxide (SiO₂),hereinafter referred to as SOS, was immersed in a solution of sulfuricacid (H₂SO₄) and hydrogen peroxide (H₂O₂) in the volume proportion of4:1 at room temperature for thirty minutes, in order to create the Si—OHgroups on the surface of the SOS, after which the sample was washed indemineralized water. Thereafter, the sample was immersed for sixtyminutes at room temperature in an anhydrous toluene solution containing5% by volume of aminopropyltritoxysilane as a silanizing agent, so as toreplace the thermodynamically stable Si—OH bonds with the much strongerSi—C ones. At the end of the incubation, the sample was placed on a hotplate at 100° C. for ten minutes. The sample was then incubated in asolution containing 150 μl of bis-succininimide suberate (a bifunctionalgroup capable of covalently binding the Bac-amp1 peptide through theN-terminal end) at a concentration of 1.6 mM dissolved in PBS solution(0.1 M pH=7.4) at 4° C. for 5 hours.

The functionalized material (SOSF) with Bac-amp1 was incubated withcultures of L. monocytogenes (3×10³ CFU/ml) in a suitable container, andthe bactericidal activity of the immobilized peptide was evaluated bymeans of vital colony counts on culture samples taken at different timeintervals. The same analysis was performed using non-functionalizedsupport (KSOS) as a control.

The data shown in FIG. 11 indicate a significant decrease of the CFU inthe presence of the functionalized material, observable after 6 hours ofincubation. The results obtained support the production of “ad hoc”functionalized materials for applications in different industrialsectors.

Example 6—Preparation of Gold Nanospheres Conjugated with IDR-Bac-Amp1

The chemical reactions leading to the formation of peptide conjugatedgold nanospheres are schematically illustrated in FIG. 12.

In details, PEG-stabilized gold nanoparticles were synthetized fromchloroauric acid (0.25 mM, 25 mL; HAuCl4 3H2O, Sigma-Aldrich, USA) bymixing poly(ethylene glycol)diacid (0.25 mM, 0.25 mL, PEG-diacid,Sigma-Aldrich, USA) and sodium tetrahydridoborate (0.01 M, 20 mL, NaBH4,Sigma-Aldrich, USA) as surfactant and reducing agent, respectively, asdescribed in Spadavecchia et al, Analyst 2014, 139, 157-164 (doi:10.1039/C3AN01794J).

The formation of the PEG-stabilized gold nanoparticles was observed asan instantaneous colour change of the solution from pale yellow tobright red after addition of the reducing agent. The reaction mixturewas centrifuged at 15.000 rpm for 30 minutes for three times and thenthe supernatant discarded. The resulting pellet of gold nanoparticleswas re-suspended in 20 ml of MilliQ-water.

As shown in FIG. 12, the carboxyl groups of PEG-stabilized goldnanoparticles obtained were covalently conjugated to the N-amine groupsof Bac-amp1 (0.125 or 0.030 mM) by reaction with1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride andN-hydroxysuccinimide (EDC 5 mM/NHS 2.5 mM, Sigma-Aldrich, USA), understirring overnight at room temperature as described in Terracciano, etal, Nanoscale 2015, 7, 20063-20074. (doi: 10.1039/C5NR05173H).

The site-specific addition of the carboxyl group of PEG at theN-terminus of the peptide was controlled by lowering the pH at which thePEGylating reaction takes place. Indeed, acid values of pH selected thereaction between PEG-COOH and the N-terminus, since its pKa is equal to2.18 while the pKa of the amino group on the side chain of lysineresidue, which could be also an attractive target for EDC/NHSconjugation chemistry, is equal to 8.95.

After the conjugation with Bac-amp1 peptide, the gold were washed twicewith MilliQ-water and re-suspended in MilliQ-water.

Example 7—Characterization of Bare and Bac-Amp1-Modified GoldNanoparticles

Transmission electron microscopy (TEM) was used to study morphology ofthe nanospheres obtained in Example 6 before and after conjugation with125 μM Bac-amp1. To this aim, 1 ml of the sample was centrifuged for 30min at 15 000 rpm and the supernatant discarded. The solid portion wasre-dispersed in 1 ml of MilliQ-water and 10 μL of NPs dispersion wereplaced on a TEM copper grid with a lacy carbon film, dried at roomtemperature and then observed by a FEI Tecnai G2 Spirit BT TEM at anaccelerating voltage of 100 KV.

Hydrodynamic diameter (size) and ζ potential (surface charge) of thenanoparticles dispersed in water (pH 7) were measured by dynamic lightscattering (DLS) using a Zetasizer Nano ZS (Malvernlnstruments, Malvern,UK) equipped with a He—Ne laser (633 nm, fixed scattering angle of 173°,room temperature 25° C.). 1 mL of NPs solution was centrifuged for 30min at 15 000 rpm and re-dispersed in MilliQ-water before eachmeasurement. Absorption spectra of bare and modified gold nanoparticles(1 mL) were recorded using a V-570 UV/VIS/NIR Cary 100 (Agilent)spectrophotometer from Jasco Int. Co. Ltd, Tokyo, Japan in the rangebetween 300 and 900 nm.

DLS was used to quantify the average diameter of bare nanoparticles in8±2 nm, while Bac-amp1-HAuNPs were 16±4. The surface charge was −26 mV,before 125 μM peptide conjugation, due to exposure of —COOH PEG groups,and +27 mV after coupling due to positive aminoacids of Bac-amp1, whichhas 4 positive net charges.

The Bac-amp1 nanoparticles colloidal solution demonstrated exceptionalchemical stability over time: the first batch was synthetized at the endof June and even if stored on bench at room temperature, it worked inexperiments in December; further control analyses (by DLS and CD)confirmed that in March size and conformational parameters did notchange.

TEM imaging showed well-dispersed nanoparticles with average size of13±5 nm (FIG. 1 (d)) and 14±7 nm (FIG. 1 (e)) after Bac-amp1 grafting;these data were in accord to that of DLS within their experimentalerrors. Despite the quite high negative surface charge and the highstability in solution, the Bac-amp1 nanoparticles revealed some tendencyto aggregate after drying. This effect was probably due to peptideclustering.

Example 8—Functionalization Yield Analysis

Reverse phase high performance liquid chromatography (RP-HPLC) was usedto analyze the yield of functionalization of the nanospheres obtained inExample 6 by means of both a direct and an indirect method.

Three different solutions were prepared for analysis.

Once functionalization was completed, the reaction mixture wascentrifuged at . . . .

The supernatant solution after centrifugation, containing the unboundpeptide, was recovered (unbound Bac-amp1). The peptide boundnanoparticles separated from the supernatant were treated with 50% (v/v)trifluoroacetic acid (TFA, Sigma-Aldrich, USA), under stirring overnightat room temperature, as described in Solid-phase peptide synthesis,Advances in Enzymology and Related Areas of Molecular Biology, Volume 32(2006): 221-296. The supernatant containing the bound peptide cleavedfrom the nanoparticles was recovered after centrifugation at 15000 rpmfor 30 min at 10° C.

Samples of the two solutions obtained above were analyzed in parallel byRP-HPLC, together with a reference solution with the initial peptideconcentration (125 μM) used for the functionalization (Bac-amp1).

For the analyses, 200 μl of sample were injected over a μBondapak C18reverse-phase column (3.9×300 mm, Waters Corporation) connected to aUFLC system (Shimadzu) using a linear gradient of 0.1% TFA inacetonitrile from 5% to 95%. All measurements were carried out intriplicate.

The absorbance curves obtained for the three samples is shown in FIG.13, which shows the absorbance curve of the reference solution(Bac-amp1), for the supernatant solution after cleavage of the peptidefrom the surface of the nanoparticles (bound Bac-amp1) and for thesupernatant solution at the end of the functionalization reaction(unbound Bac-amp1).

In the direct method, the yield of the functionalization reactionexpressed as a percentage was measured by comparing the peak areaobtained from the supernatant of the cleavage reaction with the peakarea obtained from the reference solution.

These data demonstrate that no more than 9.0±1.0% of peptide wascovalently conjugated to the gold nanoparticles surface.

In order to further validate the result, the bound peptide was alsoestimated by means of an indirect method, where the peak area of thepeptide not attached to the nanoparticles was calculated and comparedwith the peak area obtained from the reference solution. In contrast tothe direct approach, in this case the peak area quantified the amount ofthe peptide not bound to nanoparticles surface. The results demonstrateda similar trend to those obtained by direct quantification method, witha coupling yield equal to 11.0±1.0%.

On average, we therefore assumed that 10% of the initial peptideconcentration (125 μM) was covalently bound on the gold nanoparticles,resulting in approximately 12.5 μM peptide concentration in the Bac-amp1coupled nanoparticles solution.

In order to test the effect of the Bac-amp1 concentration on theimmobilization yield, which is one of the most critical parameters toachieving the formation of stable and active nanoparticles, differentamounts of peptide were tested to optimize the coupling reactionconditions. As shown in FIG. 14, it was evident that the most suitablecoupling conditions for improving the immobilization yield were obtainedwith a peptide concentration of 125 μM. Indeed, when a lesser amount ofBac-amp1 was added in the mixture, the coupling reaction resulted lesseffective as compared to 125 μM concentration of peptide and it was alsoaccompanied by precipitation of nanoparticles, probably due to the factthat the surface was not completely covered by the peptide thus favoringtheir agglomeration. Similarly, further increase in the concentrationdid not determine significant changes in the conjugation yield of thepeptide to gold nanoparticles. Hence, 125 μM was considered theconcentration of saturation and the most suitable for furtherexperimentations.

Example 9—Antimicrobial Activity of Bac-Amp1 Coupled Nanoparticles

The bactericidal efficacy of the functionalized gold nanoparticlesprepared in Example 6 was determined against L. monocytogenes (LM2food-isolated strain) and S. Typhimurium (ATCC 13311 strain) bacteria,which allowed the evaluation of the minimal bactericidal concentration(MBC50), corresponding to the lowest peptide concentration able to causeat least 50% reduction in the number of viable bacteria on agar plates.

L. monocytogenes and S. Typhimurium were grown at 37° C. in Half Fraser(Biorad-Italia) and BPW (Biomerieux-Italia) broths, respectively.

In one set of experiments, cultures with different dilutions of L.monocytogenes cells (ranging from 15×10² to 15×10⁵) or S. Typhimuriumcells (ranging from 15×10¹ to 15×10³) were incubated with a fixedconcentration of Bac-amp1 (0.16 μM) coupled to nanoparticles(Au—NPs-Bac-amp1 plates) or non-functionalized gold nanoparticles(Au—NPs plates).

Data were determined by numbering the surviving colony forming units(CFU) in the Au—NPs-Bac-amp1 plates and in the Au—NPs plates and wereexpressed as percent decrease in the number of CFU with respect to theinitial number of CFU. The error bars represent the standard deviation(SD) from the mean for a triplicate experiment (n=3).

The results obtained with L. monocytogenes bacteria are shown in FIG. 15and with S. Typhimurium cells in FIG. 16.

As can be seen from FIG. 15, Bac-amp1-loaded nanoparticles exerted astrong bactericidal activity against the food-isolate L. monocytogenesstrain LM2, causing a significant reduction in the number of CFU, ascompared to both uncoupled HAuNPs. Indeed, 99.8% and 99.9% inhibition inbacterial growth was observed in plates inoculated with 10⁴ and 10⁵bacterial concentrations, respectively, whereas the extent of inhibitionincreased up to 100% with 10² CFU/ml inoculum. In this case, theenhanced mechanism of the antimicrobial agent was also attributable toan intrinsic effect of the uncoated gold nanoparticles, which elicited96.2% inhibition in growth of gram-positive bacteria, probably due tothe release of gold ions from the nanoparticle core.

The bactericidal properties of the developed Bac-amp1 couplednanoparticles, were also tested against S. typhimurium one of the mostdangerous foodborne pathogens chosen as model of gram-negative bacteria.

As shown in FIG. 16, only the loaded nanoparticles led to a pronouncedreduction in the number of viable bacterial cells (99.91%) of S.typhimurium when 0.16 μM in the 10³ bacterial concentrations, incontrast with that observed against a lower cell density (10¹ bacterialconcentration), in which the uncoated nanoparticles exhibited 30%antimicrobial activity.

These results demonstrated that the tethered peptide after conjugationto the nanoparticles shows an excellent ability in killing bothGram-negative and Gram-positive bacteria even at infectious doses offoodborne pathogens causing outbreaks, thus suggesting a potentiallyapplication of the coupled nanoparticles in a wide array of industrialsectors. On the basis of these results, a starting inoculum ofapproximatively 15×10⁴ CFU/ml, at which no lethal activity was displayedby the bare HAuNPs, was found to be optimal for the assessment ofHAuNPs-Bac-amp1 antibacterial activity in a dose-dependent manner.

Thus, in another set of experiments, the activity of Bac-amp1 couplednanoparticles was determined incubating 15×10⁴ CFU/mL (colony formingunits/mL) of Listeria with increasing concentrations, ranging from 0.10μM to 0.58 μM, of coupled nanoparticles (Au—NPs-Bac-amp1 plates),non-functionalized gold nanoparticles (Au—NPs plates) or free peptides(Bac-amp1 plates) for 6 h at 37° C. The number of viable colonies forthe different plates at each concentration tested were counted.

As shown in FIG. 18, both Au—NPs-Bac-amp1 plates (grey bars) andBac-amp1 plates (white bars) showed a dose-dependent bacterial celldeath. However, a significantly higher killing activity, was measuredwhen the peptide was bound on gold particles.

These data demonstrate that conjugation of Bac-amp1 with goldnanoparticles not only does not disrupt the activity of theantimicrobial peptide but, surprisingly, potentiates its antimicrobialactivity.

The stability of the antimicrobial activity of the conjugatednanoparticles over time was evaluated by measuring the bactericidalactivity of 0.16 μM coupled nanoparticles against 10×10⁴ CFU/mL (colonyforming units/mL) of Listeria monocytogenes, at different timeintervals. The result obtained show that the gold tethered peptides hadan excellent long-term stability during storage and reuse, with activityremaining stable for at least 7 months. The high stability presumablyarises from two factors: the ability of nanoparticles to firmly maintainthe active peptides on their surface and the steric repulsion betweenthe peptides immobilized on the surface, which thereby prevents theaggregation and precipitation of nanoparticles. This behaviour couldhave important implications from the application point of view, due tothe possibility to recycle the conjugates multiple times without losingtheir potency.

The invention claimed is:
 1. A method for the prevention and/ortreatment of contamination of a product or a surface by Listeriamonocytogenes, the method comprising contacting the product or surfacewith a mixture comprising at least one peptide having sequenceVRLIVKVRIKRR (SEQ ID NO: 4), or a salt or solvate thereof.
 2. The methodaccording to claim 1, wherein said mixture further comprises at leastone peptide is selected from the group consisting of VRLIVAVRIWRR (SEQID NO: 1), VRLIVAVRIKRR (SEQ ID NO: 2), and VRLIVKVRIWRR (SEQ ID NO: 3),or a salt or solvate thereof.
 3. The method according to claim 1,wherein said mixture further comprises at least one peptide selectedfrom the group consisting of VRLIVAVRIWRR (SEQ ID NO: 1) andVRLIVAVRIKRR (SEQ ID NO: 2) and at least one peptide having sequenceVRLIVKVRIWRR (SEQ ID NO: 3), or a salt or solvate thereof.
 4. The methodaccording to claim 1, wherein said mixture is not in association withother bactericidal compounds.
 5. The method according to claim 1,wherein said product is a food, a material or an object.
 6. The methodaccording to claim 1, wherein said mixture is applied as a coatingcomposition on said product or surface or linked with a covalent bond tosaid product or surface.
 7. Nanoparticles comprising, covalently linkedto their surface, at least one peptide having sequence VRLIVKVRIKRR (SEQID NO: 4).
 8. The method according to claim 1, wherein said Listeriamonocytogenes is an antibiotic-resistant Listeria monocytogenesserotype.
 9. Antimicrobial composition or pharmaceutical compositioncomprising a mixture of at least one peptide selected from the groupconsisting of VRLIVAVRIWRR (SEQ ID NO: 1) and VRLIVAVRIKRR (SEQ ID NO:2) and at least one peptide having sequence VRLIVKVRIKRR (SEQ ID NO: 4).10. A product having at least one surface linked to at least one peptidehaving sequence VRLIVKVRIKRR (SEQ ID NO: 4), wherein the at least onepeptide is linked to the at least one surface by means of a covalentbond between the N-terminal amino group of the peptide and a chemicalgroup on the at least one surface.
 11. The method according to claim 1,wherein said at least one peptide having sequence VRLIVKVRIKRR (SEQ IDNO: 4) is linked to the surface of nanoparticles as claimed in claim 7.12. The product of claim 10 or the nanoparticles of claim 7, furthercomprising at least one peptide selected from the group consisting ofVRLIVAVRIWRR (SEQ ID NO: 1) and VRLIVAVRIKRR (SEQ ID NO: 2). 13.Nanoparticles as claimed in claim 7, having an average hydrodynamicdiameter measured by dynamic light scattering (DLS) comprised between 5and 90 nm.
 14. Nanoparticles as claimed in claim 7, which are hybridnanoparticles.
 15. The product of claim 10, wherein the chemical groupis selected from carboxyl groups, excited hydroxyl radicals, activatedalkoxy groups or activated aldehyde or ketone groups.
 16. Thenanoparticles of claim 7, wherein the N-terminal amino group of said atleast one peptide is covalently bound to a chemical group on saidsurface selected from carboxyl groups, excited hydroxyl radicals,activated alkoxy groups or activated aldehyde or ketone groups.
 17. Amethod for the treatment of an infection from Listeria monocytogenes ina subject in need thereof, said method comprising administering amixture comprising at least one peptide having sequence VRLIVKVRIKRR(SEQ ID NO: 4) or a pharmaceutically acceptable salt or solvate thereof.18. The method according to claim 17, wherein said Listeriamonocytogenes is an antibiotic-resistant Listeria monocytogenesserotype.
 19. The method according to claim 17, wherein said mixturefurther comprises at least one peptide selected from the groupconsisting of VRLIVAVRIWRR (SEQ ID NO: 1), VRLIVAVRIKRR (SEQ ID NO: 2)and VRLIVKVRIWRR (SEQ ID NO: 3), or a pharmaceutically acceptable saltor solvate thereof.
 20. The method according to claim 17, wherein saidat least one peptide having sequence VRLIVKVRIKRR (SEQ ID NO: 4) islinked to the surface of nanoparticles as claimed in claim 7.